Aptamers are nucleic acid species that are routinely selected in vitro through SELEX (systematic evolution of ligands by exponential enrichment). Since their introduction by the Gold and Szostak groups (Tuerk and Gold, Science, vol. 249, pp. 505-510, (1990)), aptamers have been exploited as molecular-recognition elements to detect virtually any target of interest, ranging from small molecules to proteins to even cells and tissues. Aptamers, which rival antibodies in sensitivity and specificity, are readily reproduced by chemical synthesis with low cost. Furthermore, they possess desirable storage properties and elicit little or no immunogenicity in a biological context. Owing to their advantages in comparison to antibodies, their utility in therapeutics and diagnostics has significantly expanded. Recently, the lack of inherent signaling properties of aptamers has prompted development of various strategies for transducing target-binding events into readily measurable signals for biotechnological and biomedical applications (Navani and Li, Curr. Opin. Chem. Biol., vol. 10, pp. 272-281 (2006)).
Methods that employ fluorescent reporters have proven to be particularly useful in generation of aptamer-based biosensors; these include monochromophore approaches (Jhaveri et al., J. Am. Chem. Soc., vol. 122, pp. 2469-2473 (2000)), aptamerbeacon engineering (Hamaguchi et al., Anal. Biochem., vol. 294, pp. 126-131 (2001)), structure-switching signaling (Nutiu and Li, J. Am. Chem. Soc., vol. 125, pp. 4771-4778 (2003)), in situ labeling (Merino and Weeks, J. Am. Chem. Soc., vol. 125, pp. 12370-12371 (2003)), allosteric chimeras (Wu and Curran, Nucleic Acids Res., vol. 27, pp. 1512-1516 (1999)), dye-staining approaches (Li et al., Chem. Commun., pp. 73-75 (2007)), and polymer-conjugate based fluorescent chemosensors (Ho and Leclerc, J. Am. Chem. Soc., vol. 126, pp. 1384-1387 (2004)). While these systems generally produce signals in a stoichiometric manner, attempts have been made to amplify signals by incorporation of a proximity-ligation assay (Fredriksson et al., Nature Biotechnol., vol. 20, pp. 473-477 (2002)) or an exo-nuclease-protection assay (Wang et al., Anal. Chem., vol. 76, pp. 5605-5610 (2004)) into aptamer-based sensing. Although ultrasensitive detection of proteins has been achieved, the former assay is limited to homodimer protein targets, and the latter assay suffers from tedious multistep procedures. Very recently, a DNA-polymerase assay integrated with a molecular beacon has been employed for the amplified detection of the recognition between aptamer and target small molecule (Shlyahovsky et al., J. Am. Chem. Soc., vol. 129, pp. 3814-3815 (2007)). Such techniques are in continuous demand for developing simple and easily applicable aptamer-based methods that can facilitate accurate and specific bioanalysis.